The present invention relates to compositions and methods for cloning and/or manipulating target DNA molecules at a desired sequence in a single experimental format.
In the prior art, methods for manipulation and cloning of DNA include: amplification of DNA by Polymerase Chain Reaction (PCR); cleavage of DNA with restriction endonucleases; and ligation to create recombinant molecules. Limitations of these techniques include: lack of suitable restriction sites in DNA which creates experimental difficulties in accessing a desired location of the DNA sequence for a particular manipulation; poor yield of vector compatible molecules which arises from the template-independent terminal transferase activity of polymerase which introduces a non-template nucleotide at the 3′-termini of amplified products (Clark, Nucl. Acid. Res., 16:9677-9686 (1988)); and incompatibility of the termini of the amplified fragments with termini of recipient molecules thereby preventing efficient insertion of the fragments into vectors or fusion of two or more PCR products to one another. Addition of the 3′ nucleotide as a result of terminal transferase activity of the polymerase may be overcome by end polishing of PCR products using a polymerase with 3′ to 5′ exonuclease activity (Hemsley, et al., Nucl. Acid. Res., 17:6545-6551 (1989)). Alternatively, specially prepared vectors that carry 3′-T overhangs may be used to clone PCR products carrying non-template adenine at the 3′ ends (Marchuk, et al. Nucl. Acid. Res., 19:1154 (1990)). However, both the blunt-end insertion and the T/A overhang insertion are inefficient and the above methods do not permit control over the orientation of the inserted fragment in the vector.
Efforts to improve the efficiency of blunt-end insertion of a DNA of interest by eliminating the background of self-ligated vector, include performing an insertion into a SmaI-linearized vector in the presence of SmaI restriction endonuclease which will cleave the self-ligated vector molecules at the re-generated SmaI sites (Liu, et al., BioTechniques, 12:28-30 (1992)). The PCR-Script Cloning Systems of Stratagene, Ltd. (La Jolla, Calif.) uses the rare-cleavage restriction enzyme SrfI for a similar purpose. The above methods are ineffective if the PCR product includes any internal SmaI or SrfI sites respectively, or if the site is re-generated after the insertion into the vector DNA.
Another cloning methodology involves preparing amplified segments of DNA for which different restriction sites are added to the 5′-ends of the amplification primers so as to incorporate these sites into the PCR products during amplification (Scharf, et al. Science, 233:1076-1078 (1986)). The cleavage of PCR product and vector DNA by the same restriction endonuclease produces compatible single-stranded termini that can be joined by DNA ligase. This method has several disadvantages 1) if the restriction site that is introduced into the primer is present somewhere within the PCR product, the internal site will also be cleaved during endonuclease digestion, thus, preventing cloning of full-length PCR products; 2) many restriction endonucleases inefficiently cleave sites close to the end of DNA fragment (Kaufman, et al. BioTechniques, 9:304-306 (1992)), therefore it is necessary to add 3-6 additional nucleotides to the 5′-ends of primers to ensure efficient cleavage by a particular restriction is endonuclease; 3) many restriction endonucleases are inhibited by particular components in the amplification reaction, for example, some restriction endonucleases are inhibited by single-stranded PCR primers, so an additional PCR product purification step is necessary before restriction endonuclease digestion; and 4) often restriction endonuclease generated termini are self-complementary resulting in side-products during the ligation reaction thus greatly reducing the yield of target product.
To overcome these limitations, several restriction endonuclease-free techniques have been described that allow creation of single-stranded termini on the PCR products. U.S. application Ser. No. 09/738,444 describes the use of nicking endonucleases to create single-stranded extensions that may be used specifically to join fragments with complementary ends.
Single-stranded termini complementary to the AccI and XmaI restriction endonuclease termini were generated by using the 3′ to 5′ exonuclease activity of T4 DNA Polymerase (Stoker, Nucl. Acid. Res., 18:4290 (1990)). In the presence of only dATP and dTTP, the exonuclease activity is limited to removal of only G and C nucleotides, thus creating the requisite single-stranded termini for sub-cloning into a AccI- and XmaI-cleaved plasmid vector. In technology referred to as Ligation-Independent Cloning of PCR products (LIC-PCR) (Aslanidis, et al., Nucl. Acid. Res., 18:6069-6074 (1990)) target DNA is amplified with primers containing 12 additional nucleotides at their 5′ ends that lack cytosine. As a result, the PCR product on the 3′ ends is flanked by a 12-nucleotide sequence lacking guanine. Treatment of the PCR product with the 3′ to 5′ exonuclease associated with T4 DNA Polymerase in the presence of dGTP removes the 3′ terminal sequences until the first dGMP residue is reached, thus leaving a 12 nucleotide 5′ single-stranded extension. However disadvantages of this technology include the need for a special vector having compatible 12 nucleotide 5′ single-stranded extensions for cloning. The preparation of such vectors include amplification of the entire vector with primers containing 12 nucleotide tails complementary to the tails used for amplification of target fragment and subsequently treating the amplified vector with T4 DNA Polymerase to create complementary 12 nucleotide long single-stranded extensions. A modified technique of LIC-PCR has been described, where the specific sequences devoid of particular bases are engineered into plasmid vectors to replace the vector amplification step by a restriction digestion step (Haun, et al. BioTechniques, 13:515-518 (1992); Kuijper, et al. Gene, 112:147-155 (1992); Cooney, BioTechniques, 24:30-33 (1998)).
A disadvantage of the above-described methods is the need to remove leftover dNTP, before subjecting the PCR product to exonuclease treatment. The use of this technology is limited to sequences devoid of at least one nucleotide, and in addition the use of non-specific exonucleases to manipulate DNA may give rise to sequence rearrangements at the position of vector-product junction of recovered recombinant molecules.
Single-stranded overhangs or extensions have been produced during PCR by incorporating the non-base residue, 1,3-propanediol, into primer sequences. This has the effect of terminating DNA synthesis (Kaluz, S. et al. (1994) Nucl.Acid.Res., 22, 4845). During PCR, Taq DNA Polymerase stops at the non-replicable element, leaving a portion of the primer as a single strand. Since 1,3-propanediol also inhibits DNA replication processes in vivo, the repair machinery of the bacterial host has to remove the non-replicable element potentially causing unwanted sequence rearrangements in the recovered recombinant molecules.
Cloning and manipulating genes with the use of a DNA repair enzyme, Uracil DNA Glycosylase (UDG), has been described. (Rashtchian et al. U.S. Pat. No. 5,137,814; Berninger, U.S. Pat. No. 5,229,283; Nisson, et al., PCR Methods & Applications, 1:120-123 (1991); Rashtchian, et al., PCR Methods & Applications, 2:124-130 (1992).; Booth, et al., Gene, 146:303-308 (1994); Rashtchian, Current Biology, 6:30-36 (1995)) UDG recognizes uracil lesions in single- or double-stranded DNA and cleaves the N-glycosylic bond between the deoxyribose moiety and the base leaving an abasic site. During PCR, Taq DNA Polymerase inserts deoxyadenisine opposite a deoxyuridine (U) lesion. Target DNA and cloning vectors can be amplified with primers at the 5′ ends carrying dUMP-containing tails. Subsequent treatment with the UDG glycosylase results in formation of multiple abasic sites on the ends of the amplified product. Strand separation across the modified portion of the amplified product and the vector that contains complementary ends (generated by the same approach) provides a re-annealed recombinant product having protruding single-stranded flaps which should be removed in vivo by the repair machinery of the bacterial host. Cloning of cDNAs by single-primer amplification (SPA) that employs a dU-containing primer has been described in U.S. Pat. No. 5,334,515.
Since UDG does not cleave the phosphodiester backbone, the efficiency of strand separation to a great extent depends on the number of dUMP residues within the 5′ ends of PCR products. Hence, at least one third of the 5′ tails of the PCR primer should consist of dUMP to achieve efficient strand separation between two strands of DNA duplex (U.S. Pat. Nos. 5,137,814 and 5,229,283). Another disadvantage of this method is that the entire plasmid vector must be amplified by PCR to produce the linear vector flanked by the complementary extensions suitable for sub-cloning of the UDG-treated PCR fragments.
UDG glycosylase has also been used to create SacI restriction endonuclease-like cohesive ends on PCR fragments which are suitable for cloning into SacI-linearized vectors (Smith, et al. PCR Methods & Applications, 2:328-332 (1992); Watson, et al. BioTechniques, 23:858-862 (1997)). However, this technology is very limited, as it allows cloning of PCR amplified product only into a Sacd site. Another disadvantage of this method is that SacI-like cohesive termini are self-complementary. Therefore a variety of unwanted side-products are generated upon ligation thus reducing the use of this technology in DNA manipulations other than cloning of PCR products.
A chemical method for creating single-stranded overhangs on PCR products employs PCR primers containing ribonucleotides, such as rUMP or rCMP (Chen, et al. BioTechniques, 32:517-520 (2002); Jarell, et al. U.S. Pat. No. 6,358,712). After amplification, the PCR products are treated with rare-earth metal ions, such as La3+ or Lu3+ (Chen, et al. BioTechniques, 32:517-520 (2002)) or sodium hydroxide (Jarell, et al. U.S. Pat. No. 6,358,7112) to hydrolyze the phosphodiester bond between the deoxyribonucleotide and the ribonucleotide. Disadvantages include the high cost of PCR primers and in addition the vector DNA must be prepared by PCR with the use of primers containing ribonucleotides to generate compatible termini suitable for sub-cloning.
Some of the PCR-based sub-cloning techniques described above can also be used for site-specific DNA mutagenesis. However their application is limited to cases in which, it is possible to introduce a specific change without disrupting the rest of the coding sequence. For example, when suitable restriction sites are located in close proximity to the nucleotide sequence targeted for mutation, the PCR-based oligonucleotide-directed site-specific mutagenesis is routinely used to introduce desired mutations into target DNA sequences and the mutated PCR fragment is then introduced in place of the wild-type sequence using restriction endonuclease digestion (Higuchi, et al. Nucl. Acid. Res., 16:7351-7367 (1988)). However, when the appropriate naturally occurring restriction sites are not available, additional experimental procedures must be performed to introduce internal changes.
Another PCR-dependent mutagenesis method uses a “megaprimer”. Megaprimers are long, double-stranded DNAs which are often difficult to denature, to anneal and to extend to a full-length product. Consequently, the method has been found to be problematic when the megaprimer is longer than several hundred base pairs (Kammann, et al. Nucl. Acid. Res., 17:5404 (1989); Sarkar, et al. BioTechniques, 8:404-407 (1990); Sarkar, et al. Nucl. Acid. Res., 20:4937-4938 (1992); Landt, et al. Gene, 96:125-128 (1990); Ling, et al. Analytical Biochemistry, 254:157-178 (1997); Smith, et al. BioTechniques, 22:438-442 (1997); Colosimo, et al. BioTechniques, 26:870-873 (1999)).
Another PCR-dependent site-directed mutagenesis technique referred to as “overlap-extension” PCR has been described (Higuchi, et al. Nucl. Acid. Res., 16:7351-7367 (1988); Ho, et al. Gene, 77:51-59 (1989)). Two primary PCR reactions produce two overlapping DNA fragments, both bearing the same mutation introduced via mutagenic primers in the region of the overlap sequence. These fragments are then combined, denatured and re-annealed to generate a hetero-duplex product via the overlapping sequence. In the re-annealed hetero-duplex product, the 3′ overlap of each strand serves as a primer for the extension of the complementary strand. The extended full-size fusion is then amplified in a second round of PCR using the outside primers. The overlap-extension method is laborious and inefficient in many practical applications for several reasons: it requires the purification of intermediate PCR product to remove mutagenic primers; it requires two full rounds of PCR, which increases the possibility of introducing the undesired mutations; and the efficiency of annealing heterologous molecules across the overlap region is greatly reduced by the presence of a full-length complementary strand of either fragment.
No existing single PCR-based method for site-directed mutagenesis and cloning appears to solve all of the problems associated with in vitro DNA manipulations. A single strategy for achieving a variety of DNA manipulations, such as linking, adding, deleting or changing nucleotide segments at any desired location of target DNA molecule would be desirable.